Immunoselection is a generic term that encompasses a variety of techniques in which the specificity of a selection system is conferred by an antibody or an antibody-like molecule such as a lectin or hapten. An example of such specificity is the affinity of an antibody for a specific cell surface antigen. Two general types of immunoselection techniques are practiced. Negative immunoselection involves the elimination of a specific subpopulation of components from a heterogeneous population such as a suspension of various cell types. Exclusively negative selection techniques have an inherent disadvantage: Although specific component types can be removed, the remaining components are an enriched but not a pure population. In contrast, positive immunoselection refers to the direct selection and recovery of a specific component, such as cells which express a given specificity from among a heterogeneous group of contaminating cell types. Rather than eliminating the undesired elements, positive immunoselection techniques can lead directly to the selective enrichment and even purification of targeted antigen-bearing cells. Another difference between the two techniques is that viable, functional cells are typically the desired end product of positive immunoselection techniques, while the continuing viability of negatively selected cells is usually not required.
Bone marrow transplantation provides an illustrative example of a clinical application in which immunoselection techniques have shown promise. Bone marrow transplantation is being utilized for treatment of increasing numbers of patients with hematological malignancies, aplastic anemia, solid tumors, certain congenital hematological disorders, and immunodeficiency states. The basic aim of the bone marrow transplantation treatment is to replace a defective component of the blood or immune system by first destroying the defective cells in the patient (recipient) and then transplanting omnipotential, hematopoietic stem cells derived from either the recipient (autologous transplant) or another donor (allogenic transplant). The transplanted hematopoietic stem cells can potentially differentiate into normal cell types that will replace the defective cells. Unfortunately, about seventy percent of patients with aplastic anemia and even fewer with leukemia survive more than one year following the bone marrow transplantation procedure. The major obstacles that limit the success of this procedure include graft-versus-host disease in patients given allogeneic transplants, and relapse possibly caused by the presence of residual tumor cells following autologous marrow transplants.
Investigators have demonstrated that graft-versus-host disease can be prevented in animals by removing T cells from the donor marrow before transplantation into the recipient. Similarly, successful autologous marrow transplants have been performed in animals in which tumor cells were first removed in vitro from the donor marrow before transplantation into the recipient. The successes of such animal studies have stimulated the development of various in vitro methods designed to eliminate T cells or tumor cells from human bone marrow. These methods have included the use of antibodies with complement, antibody-toxin conjugates, various physical separation or fractionation procedures, chemotherapeutic agents, and magnetic immunoselection. Another approach has been to use a chromatography column made of gel substrate to which monoclonal antibodies directed against T cells or tumor cells are directly linked. Transplantation 38:136, 1984; Proceedings, American Society of Clinical Oncology, Toronto 3:269 (Abstract #C-1052), 1984. Such a negative immunoselection technique avoids the toxicity to hematopoietic stem cells and decreased recovery of bone marrow that characterize some of the other in vitro techniques. However, the large quantities of monoclonal antibodies required for direct attachment to the gel (5 mg/ml) has made scale up of that procedure for clinical application to human bone marrow transplantation impractical.
The high binding efficiency between avidin and biotin has also been applied in prior immunoselection techniques. Biotin is a vitamin present in minute amounts in every living cell. Avidin is a glycoprotein isolated from raw egg white. Biotin selectively combines with avidin with a remarkably high affinity constant (K.sub.m =10.sup.-15 M). The avidin-biotin complex has been used as a tool in molecular biology for the following objectives: isolation of biotin-derivatized materials by affinity chromatography; affinity labeling and identification studies; affinity cytochemical labeling for localization studies in fluorescence and electron microscopy; inhibition of bacteriophage; and study of cell surface molecular interactions. Bayer and Wilcheck, TIBS, N257-N259, November 1978. With regard to the isolation of biotin-derivatized materials by affinity chromatography, biotinized rat thymocytes were reportedly retrieved using avidin which was covalently coupled to nylon meshes; no attempts were made to remove the cells from the avidin. Exp.Cell Res. 100:213-217, 1976. T-cells were reportedly depleted by treating spleen cells with biotin-conjugated antibody directed to T-cell antigen, followed by panning twice on avidin-coated plates. J.Exp.Med. 159:463-478, 1984. Another negative immunoselection technique employing avidin-biotin is described in U.S. Pat. No. 4,298,685.
Biotinylated antibodies have been used to label cells which were then removed by passing them over immobilized avidin, but unfortunately the binding between avidin and biotin has proved to be essentially irreversible. In order to use this technique for positive immunoselection, antibodies have been conjugated with biotin analogs that reportedly have the advantage that they adhere to avidin containing matrices but can be displaced by washing with authentic biotin. J.Immunol.Meth. 56:269-280, 1983.
Biotinylated antibodies and avidin-coupled sheep erythrocytes were used for rosette formation, followed by separating rosetting cells from non-rosetting cells on a density gradient. This method was reported to present the possibility of obtaining both positive and negative subpopulations with high yield. J.Immunol.Meth. 67:389-394, 1984. However, recovered cells are coated with sheep erythrocytes which could potentially affect the function of the rosetted cell population.
U.S. Pat. Nos. 4,253,996 and 4,276,206 note that the application of insolubilized avidin in the affinity chromatographic isolation of biotin-containing molecules has been limited due to the problem in recovery since the affinity of avidin to biotin is so high. Both patents reportedly circumvent this problem by preparing avidin-Sepharose conjugates with reduced affinity for biotin and thereby reportedly provide an operable positive selection technique using the avidin-biotin system.
Uses of a second, anti-species antibody in conjunction with immunoassays employing avidin and biotin are disclosed in U.S. Pat. Nos. 4,228,237, 4,468,470, and 4,496,654.
It would be advantageous to provide an improved negative immuno-selection method whereby specific subpopulations of cells such as T cells and/or tumor cells could be removed from heterogeneous cell populations such as bone marrow cells with a high degree of selectivity and with a low nonspecificity, using relatively small amounts of antibody and minimal manipulation of the nonselected cells. It would also be desirable to provide an improved positive immunoselection system.